Many biological substances, especially nucleic acids, present special challenges in terms of isolating them from their natural environment. On the one hand, they are often present in very small concentrations and, on the other hand, they are often found in the presence of many other solid and dissolved substances, e.g., after lysis of cells. This makes them difficult to isolate or to measure, in particular in biospecific assays which allow the detection of specific nucleic acids or the detection of specific properties of a nucleic acid. Such biospecific assays play a major role in the field of diagnostics and bioanalytics in research and development. Examples of biospecific assays are hybridization assays, immunoassays, and receptor-ligand assays. Hybridization assays use specific base-pairing for the molecular detection of nucleic acid analytes, e.g., RNA and DNA. Hence, oligonucleotide probes with a length of 18 to 20 nucleotides may enable the specific recognition of a selected complementary sequence, e.g., in the human genome. Another assay which entails the selective binding of two oligonucleotide primers is the polymerase chain reaction (PCR) described in U.S. Pat. No. 4,683,195. This method allows the selective amplification of a specific nucleic acid region to detectable levels by a thermostable polymerase in the presence of deoxynucleotide triphosphates in several cycles.
As described above, before nucleic acids may be analyzed in one of the above-mentioned assays or used for other processes, they have to be isolated or purified from biological samples containing complex mixtures of different components such as, e.g., proteinaceous and non-proteinaceous components. Often, for the first steps, processes are used which allow the enrichment of the component of interest, i.e., the nucleic acids. Frequently, these are contained in a bacterial cell, a fungal cell, a viral particle, or the cell of a more complex organism, such as a human blood cell or a plant cell. Nucleic acids as a component of interest can also be called a “target component”.
To release the contents of said cells or particles, they may be treated with enzymes or with chemicals to dissolve, degrade, or denature the cellular walls and cellular membranes of such organisms. This process is commonly referred to as lysis. The resulting solution containing such lysed material is referred to as a lysate. A problem often encountered during the lysis is that other enzymes degrading the target component, e.g., deoxyribonucleases or ribonucleases degrading nucleic acids, come into contact with the target component during lysis. These degrading enzymes may also be present outside the cells or may have been spatially separated in different cellular compartments before the lysis and come now into contact with the target component. Other components released during this process may be, e.g., endotoxins belonging to the family of lipopolysaccharides which are toxic to cells and can cause problems for products intended to be used in human or animal therapy.
In the next steps of the sample preparation which follow on the lysis step, the nucleic acids are further enriched. Nucleic acids are normally extracted from the complex lysis mixtures before they are used in a probe-based assay. There are several methods for the extraction of nucleic acids. Sequence-dependent or biospecific methods include, e.g., affinity chromatography or hybridization to immobilized probes. Sequence-independent or physico-chemical methods include, e.g., liquid-liquid extraction with phenol-chloroform, precipitation with pure ethanol or isopropanol, extraction with filter paper, extraction with micelle-forming agents as cetyl trimethyl ammonium bromide, binding to immobilized, intercalating dyes such as acridine derivatives, adsorption to substrates such as silica gel or diatomic earths, and adsorption to magnetically attractable glass particles or organo silane particles under chaotropic conditions. Direct binding of the nucleic acids to a substrate such as a material with a silica surface is preferred because among other reasons the nucleic acids do not have to be modified, and even native nucleic acids can be bound.
Particularly interesting for extraction purposes is the adsorption of nucleic acids to a glass surface, although other surfaces are possible.
Many procedures for isolating nucleic acids from their natural environment have been proposed in recent years by the use of their binding behavior to substrates such as glass surfaces. It is common to use chaotropic agents such as, e.g., guanidine thiocyanate or anionic, cationic, zwitterionic, or non-ionic detergents when nucleic acids are intended to be set free. It is also an advantage to use proteases, which rapidly degrade these enzymes or unwanted proteins. Nucleic acids which are set free, e.g., after cell lysis and/or lysis of cellular organelles such as mitochondria, plastids, nuclei, or other nucleic acid-containing organelles, can be purified by way of binding to a substrate such as a mineral support, washing said mineral support with the bound nucleic acids, and releasing, i.e., desorbing said nucleic acids from said mineral support. For a washing step, conditions are chosen by the skilled artisan under which the nucleic acids remain adsorbed to the mineral support. Typically, greater than 40%, more typically greater than 50%, more typically greater than 70%, more typically greater than 80%, even more typically greater than 90%, even more typically greater than 95%, even more typically greater than 99% of the nucleic acids remain adsorbed to the mineral support. For the desorbing step, conditions are chosen by the skilled artisan under which the nucleic acids are released from the mineral support. Typically, greater than 40%, more typically greater than 50%, more typically greater than 70%, more typically greater than 80%, even more typically greater than 90%, even more typically greater than 95%, even more typically greater than 99% of the nucleic acids are released from the mineral support.
Adsorption of nucleic acids to glass particles or silica particles in the presence of chaotropic salts is known to the art (Vogelstein, B., and Gillespie, D., Proc. Natl. Acad. Sci. USA 76 (1979) 615-619) and provides the basis for chromatographic purification and separation processes for nucleic acids. Also known to the art are methods to isolate and purify RNA and DNA from lysates using high concentrations of chaotropic salts, e.g., sodium iodide, sodium perchlorate, and guanidine thiocyanate (Boom, R., et al., J. Clin. Microbiol. 28 (1990) 495-503; Yamada, O., et al., J. Virol. Methods 27 (1990) 203-209). The purification of plasmid DNA from bacteria on glass dust in the presence of sodium perchlorate is described in Marko, M. A., et al., Anal. Biochem. 121 (1982) 382-387. In DE 37 24 442, the isolation of single-stranded M13 phage DNA on glass fiber filters by precipitating phage particles using acetic acid and lysis of the phage particles with perchlorate is described. The nucleic acids bound to the glass fiber filters are washed and then eluted with a methanol-containing tris/EDTA buffer. A similar procedure for purifying DNA from lambda phages is described in Jakobi, R., et al., Anal. Biochem. 175 (1988) 196-201. The procedure entails the selective binding of nucleic acids to glass surfaces in chaotropic salt solutions and separating the nucleic acids from contaminants such as agarose, proteins, or cell residue. To separate the glass particles from the contaminants, the particles may be either centrifuged or fluids are drawn through glass fiber filters. This is a limiting step, however, that prevents the procedure from being used to process large quantities of samples.
The use of magnetic particles to immobilize nucleic acids after precipitation by adding salt and ethanol is more advantageous and is described, e.g., in Alderton, R. P., et al., Anal. Biochem. 201 (1992) 166-169 and WO 91/00212. In this procedure, the nucleic acids are agglutinated along with the magnetic particles. The agglutinate is separated from the original solvent by applying a magnetic field and performiing a wash step. After one wash step, the nucleic acids are dissolved in a tris buffer. This procedure has a disadvantage, however, in that the precipitation is not selective for nucleic acids. Rather, a variety of solid and dissolved substances are agglutinated as well. As a result, this procedure can not be used to remove significant quantities of any inhibitors of specific enzymatic reactions that may be present. Magnetic porous glass is also available on the market that contains magnetic particles in a porous, particular glass matrix and is covered with a layer containing streptavidin. This product can be used to isolate biological materials, e.g., proteins or nucleic acids, if they are modified in a complex preparation step so that they bind covalently to biotin. Magnetizable particular adsorbents proved to be very efficient and suitable for automatic sample preparation. Ferrimagnetic and ferromagnetic as well as superparamagnetic pigments are used for this purpose. The most typical magnetic glass particles are those described in WO 01/37291.
Purification of a nucleic acid by way of adsorbing the same to a substrate such as a mineral substrate in the presence of high concentration of salts is also applied to other complex mixtures. Examples therefor are known to the person skilled in the art of molecular biology and include reaction mixtures following, e.g., in vitro synthesis of nucleic acids such as PCR, restriction enzyme digestions, ligation reactions, etc.. In Vogelstein, B., and Gillespie, D., Proc. Natl. Acad. Sci. USA 76 (1979) 615-619, for instance, a procedure for binding nucleic acids from agarose gels in the presence of sodium iodide to ground flint glass is proposed. Another application for purification of a nucleic acid by way of adsorbing the same to a substrate such as a mineral substrate in the presence of a high concentration of salts is the removal of pyrogenic contaminants which may have copurified with the nucleic acid.
The mechanism by which nucleic acids bind to the mineral support in the presence of chaotropic agents is not entirely clear. It is hypothesized that the interaction between the nucleic acids and the solvent is influenced such that the nucleic acids adsorb to the mineral support and denaturant. In the presence of high concentrations of chaotropic agents the reaction is almost quantitative. The adsorbed nucleic acids can be eluted by applying to the mineral support buffers of low ionic strength.
EP 0 658 164 describes a method for the chromatographic purification of nucleic acids by way of chromatographic purification. Nucleic acids are adsorbed to a substrate, i.e., a mineral support, from an aqueous adsorption solution with a high salt concentration which typically contains a chaotropic agent. The aqueous adsorption solution comprises 1%-50% of aliphatic alcohol with a chain length of C1-C5 and/or polyethylene glycol and/or hydrophobic inorganic and/or organic polymers and/or organic acid such as trichloroacetic acid.
The methods for the isolation/purification of nucleic acids of the state of the art have certain disadvantages. Such disadvantages relate to, e.g., purity, selectivity, recovery rate, laboratory safety, and convenience, as well as to the speed of the isolation/purification process. For example, in protocols using a phenol/chloroform extraction, residual phenol is often a problem for certain post isolation procedures, particularly for enzymatic reactions such as a digestion with a restriction enzyme, the polymerase chain reaction (PCR), or a ligase-mediated reaction. Generally, elevated concentrations of residual reagents from the purification/isolation process may pose a problem. It is therefore desired to keep residual amounts of the reagents used during the purification procedure as low as possible in the purified nucleic acid. Another potential problem related to purity is the coextraction of certain substances from the adsorption matrix (leaching). It is therefore desired to keep residual amounts of compounds liberated during the purification procedure by leaching as low as possible in the purified nucleic acid.
Another disadvantage of state of the art protocols which use ethanol or isopropanol in the adsorption solution is the high volatility and flammability of such alcohols. On the one hand, these flammable alcohols are potential hazards in laboratory practice. Also, depending on national regulations, flammable alcohols may pose logistical problems with regard to allowable storage and transport. In addition, volatile alcohols are difficult to dose with precision because of their vapor pressure. It is therefore desired to replace flammable alcohols by substances which are less hazardous or/and which pose fewer logistical problems.
Exemplary kits which are commercially available for sample preparation of nucleic acids are the High Pure product line (Roche Diagnostics GmbH, Mannheim, Germany). The adsorption solution is transferred to a High Pure column and passed through a fleece containing glass fiber material. During this process the nucleic acids are adsorbed to the glass material. When using the columns of the Roche High Pure kit and a protocol for nucleic acid isolation/purification from serum making use of ethanol in the adsorption solution, it was noted that high triglyceride concentrations in serum lead to a prolonged time needed to pass the adsorption solution through the glass fiber fleece (also see Example 6). It is therefore desired to identify a substitute for ethanol which, considering sample preparation from serum with high triglyceride concentrations, reduces the time needed to pass the adsorption solution through the glass fiber fleece.
The problem underlying the present invention is therefore to provide an alternative method for the purification of a nucleic acid using alternative substances in the aqueous adsorption solution in order to facilitate the binding of a nucleic acid to a substrate such as a mineral support.